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Uncovering an unfolded protein response in Trypanosoma brucei by a computational approach Shai Carmi Bar-Ilan University Deptartment of physics and the faculty of life sciences June 2009 The endoplasmic reticulum (ER) The ER is a membrane enclosed Eukaryotic organelle that provides an environment in which secreted or other proteins with hydrophobic domains can be properly folded and targeted without aggregating. Responsible for protein •Folding •Modification •Transport •Degradation •Lipid and steroid synthesis •Calcium storage The unfolded protein response (UPR) ER stress in the form of high concentration of misfolded proteins triggers the unfolded protein response: A signaling pathway which relieves stress by selective gene upregulation and other mechanisms. Prolonged ER stress leads to programmed cell death (Apoptosis). Metazoan UPR nonconventional splicing From Allen Volchuk’s lab website UPR in protozoans (such as yeast) has only the IRE1-HAC1 system. UPR sensors and signal transduction The three ER stress sensors respond differently to high concentration of unfolded proteins: Bernales et al., 2006 IRE1 oligomerizes, autophosphrylates and activates its RNase domain to remove an intron from HAC1 mRNA to facilitate its translation. hac1p then binds to specific promoters to activate transcription of UPR genes. PERK phosphorylates eIF2α to block the formation of ribosomal preinitiation complex and to attenuate translation, while at the same time preferentially translate the ATF4 transcription factor. ATF6 migrates to the Golgi upon UPR induction, where it is cleaved and activates transcription from Metazoan Xbp1 nonconventional splicing other promoters. results in frame shift and activation. UPR components The ultimate goal of the response to decrease the concentration of misfolded proteins in the ER. Upregulate chaperones that increase the chances of proper folding by preventing aggregation and covalent modification. Express lipid biosynthesis genes to expand the ER. Remove proteins from the ER by accelerated degradation and transportation out of the ER. Relieve ER load by global translation attenuation and specific mRNA degradation of ER client proteins. Genes which participate in other processes (e.g., protection from oxidative stress, signaling, and metabolism) are also upregulated. More facts about the UPR The three arms of UPR induction are not just independent on/off switches but are modulated by interconnections and external factors. ER stress can be artificial (‘pharmacological’) or biological. The entire UPR or selected branches are induced under normal physiological conditions (development of B cells into ‘antibody factories’ plasma cells, nutrient deprivation) as well as in disease: diabetes, viral infection, retinal degeneration, cancer (hypoxia). All three branches also upregulate genes that elicit programmed cell death after prolonged stress via standard pathways (e.g. calcium release from the ER, cytochrome c release, caspase activation, dephosphorylation of eIF2α, reactive oxygen species production). Pro-survival and pro-apoptotic pathways compete. For example: PERK also phosphorylates eIF2α but at the same time upregulates CHOP and consequently GADD34 which dephosphorylates eIF2α. Trypanosoma brucei Trypanosomes are parasitic eukaryotes that diverged 200-500 million years ago. Pathogens of the African Sleeping sickness. Live in the gut of the Tsetse fly (the “procyclic form”), then transfer to the bloodstream of humans and cattle. Have unique biology: - Kinetoplast - RNA editing with gRNA - trans-splicing - Variable surface glycoproteins From Mark Field’s lab website . S.C mRNA processing mRNA is processed by trans-splicing and polyadenylation. T. brucei genes have no promoters. Gene expression is regulated by controlling splicing, mRNA stability, and translation. Gene1 Gene2 Gene3 Gene4 Polycistronic Transcript SL Trans-Splicing= And Polyadenylation= AAAA AAAA AAAA AAAA Itai Dov Tkacz Unfolded protein response? No homolouge of IRE1, the ER stress sensor, was identified to date in T. brucei. T. brucei cannot regulate gene expression by transcription activators. Does T. brucei have unfolded protein response and how is it executed? Applying ER stress Silencing the translocon components. Ribosome Treating with the reducing agent Dithiothreitol (DTT). mRNA ER membrane © Saggit Goldshmidt Sec63 After translation initiation, proteins processed by the ER are attached to the ER membrane by the signal recognition particle (SRP), after which they are transported into the ER lumen through a channel called the translocon. DTT reduces disulfide bonds (S-S) which are essential for the proper folding of many proteins. Leads to UPR in various organisms. SL-RNA Silencing (SLS) Lustig et al., EMBO reports, 2007 SLS induction Nucleoplasm 42 42 50 26 ? SL 42 42 SL RNA 42 SL DNA SL 42 Itai Dov Tkacz. No trans-splicing 42 No mRNA No protein Increase in levels of the chaperone BiP is also obsreved. Cell death SL-RNA is the only gene whose expression is controlled by interaction between a promoter and an activator- the tSNAP complex. 4mM DTT min 0 60 120 SL SR1 . Anat Kabi, 2007 How does SLS compare to classic UPR in other organisms? How is the ER stress signal transmitted into the nucleus? 240 Microarrays Microarrays are chips on which thousands of DNA oligos are printed in an array. Each oligo represents a fragment of one gene. Total RNA is isolated and then reverse transcribed to cDNA, during which it is also labeled with fluorescent dye. The cDNA is hybridized with the array after which the array is read by a scanner. The higher the concentration of a given gene, the brighter will be the corresponding spot in the array. Expression profiles of entire genomes can be obtained within a single experiment. Other uses include ChIP or construction of specialized arrays to detect alternative splicing. Wikipedia The transcriptome of ER stressed cells Use microarrays to track the change in mRNA levels of stressed cells in comparison to untreated cells. DTT r=0.85 Every gene is a dot Every gene is a line Arrays for 1 hour and 3 hours are consistent. Regulation exists: many genes are up and downregulated. Functional analysis of regulated genes Genes upregulated at least 1.5-fold after both 1 hour and 3 hours DTT treatment were classified to functional categories. No filtering can be done as in other organisms. The class with the largest number of upregulated genes is the secretory pathway. Many signaling genes were upregulated, suggesting the process is regulated. Genes related to transport, metabolism, gene expression, mitochondria, and cell cycle were also upregulated. We also observed upregulation in DNA repair, oxidative stress, and calcium homeostasis genes, as expected in cells committing apoptosis; and in motility genes, raising the hypothesis that trypanosomes have evolved a ‘chemotaxis’-like mechanism. Functional analysis of regulated genes Upregulated genes in T. brucei belong to similar functional categories as UPR genes from other organisms. Hanoch Goldshmidt, S.C., 2009 Genes downregulated after 1 hour treatment with DTT are enriched with ER client proteins (P<10-4). ER stress leads to programmed cell death Loss of mitochondrial membrane potential Production of reactive oxygen species DNA ladderinggenomic DNA digested at specific intervals Concentration of cytoplasmic calcium ions increases after treatment also indicating apoptosis Also observed endoG release Propidium iodide (PI)- correlates with membrane permeability. Annexin V binds to Phosphatidylserine (PS). Exposed PS indicates apoptosis. Hanoch Goldshmidt, Dvorah Mattas, 2009 How to identify the genes which mediate SLS? The transcriptomes of DTT treatment and Sec63 silencing are quite dissimilar even though both lead to SLS. The few commonly upregulated genes may participate in the SLS pathway. Choose two candidate genes for further experimental analysis. Candidates Tb927.1.2100Caplain-like cysteine peptidase. Calcium binding protein. Lost its proteolytic activity. Membranal, not essential. Role in apoptosis signaling? . S.C Ephrat ben-Mayor Tb10.6k15.1520small GTPase. Homolog of human RagC. Homolog participates in amino-acid starvation signaling to mTOR and localizes in the nucleus and the cytoplasm. Essential in T. brucei. Experimentally test candidate genes Prepare double silenced cell line where both the candidate gene and Sec63 are silenced by RNAi. Test for SLS by northern blot. Silencing is induced by Tetracyclin. Conclusions T. brucei respond to ER stress by upregulating specific genes in a pattern that resembles the response of other organisms. In addition, mRNA metabolism is arrested in synergy with programmed cell death that takes place at later times. We conclude that T. brucei has UPR with trypanposome specific elements. Computational approach suggests proteins that participate in the signaling pathway. More experimental work is required to establish a concrete statement. Special thanks to all Michaeli’s lab members: Ephrat ben-Mayor Hanoch Goldshmidt Nasreen Hag-Yahia Ronen Hope Sachin Kumar Gupta Asher Pivko Mali Romano Itay Dov Tkacz Pawel Tuliniski Damian Visnovezky Vadim Volcov Liat Wulffhart Dr. Devorah Mattas Dr. Chaim Wachtel Prof. Shulamit Michaeli